Aptamers are nucleic acid molecules having specific binding affinity to molecules through interactions other than classic Watson-Crick base pairing.
Aptamers, like peptides generated by phage display or monoclonal antibodies (MAbs), are capable of specifically binding to selected targets and, through binding, block their targets' ability to function. Created by an in vitro selection process from pools of random sequence oligonucleotides (FIG. 1), aptamers have been generated for over 100 proteins including growth factors, transcription factors, enzymes, immunoglobulins, and receptors. A typical aptamer is 10-15 kDa in size (30-45 nucleotides), binds its target with sub-nanomolar affinity, and discriminates against closely related targets (e.g., will typically not bind other proteins from the same gene family). A series of structural studies have shown that aptamers are capable of using the same types of binding interactions (hydrogen bonding, electrostatic complementarity, hydrophobic contacts, steric exclusion, etc.) that drive affinity and specificity in antibody-antigen complexes.
Aptamers have a number of desirable characteristics for use as therapeutics including high specificity and affinity, biological efficacy, and excellent pharmacokinetic properties. In addition, they offer specific competitive advantages over antibodies and other protein biologics, for example:
1) Speed and control. Aptamers are produced by an entirely in vitro process, allowing for the rapid generation of initial therapeutic leads. In vitro selection allows the specificity and affinity of the aptamer to be tightly controlled and allows the generation of leads against both toxic and non-immunogenic targets.
2) Toxicity and Immunogenicity. Aptamers as a class have demonstrated little or no toxicity or immunogenicity. In chronic dosing of rats or woodchucks with high levels of aptamer (10 mg/kg daily for 90 days), no toxicity is observed by any clinical, cellular, or biochemical measure. Whereas the efficacy of many monoclonal antibodies can be severely limited by immune response to antibodies themselves, it is extremely difficult to elicit antibodies to aptamers (most likely because aptamers cannot be presented by T-cells via the MHC and the immune response is generally trained not to recognize nucleic acid fragments).
3) Administration. Whereas all currently approved antibody therapeutics are administered by intravenous infusion (typically over 2-4 hours), aptamers can be administered by subcutaneous injection. This difference is primarily due to the comparatively low solubility and thus large volumes are necessary for most therapeutic MAbs. With good solubility (>150 mg/ml) and comparatively low molecular weight (aptamer: 10-50 KD; antibody: 150 KD), a weekly dose of aptamer may be delivered by injection in a volume of less than 0.5 ml. Aptamer bioavailability via subcutaneous administration is >80% in monkey studies (Tucker, 1999). In addition, the small size of aptamers allows them to penetrate into areas of conformational constrictions that do not allow for antibodies or antibody fragments to penetrate, presenting yet another advantage of aptamer-based therapeutics or prophylaxis.
4) Scalability and cost. Therapeutic aptamers are chemically synthesized and consequently can be readily scaled as needed to meet production demand. Whereas difficulties in scaling production are currently limiting the availability of some biologics and the capital cost of a large-scale protein production plant is enormous, a single large-scale synthesizer can produce upwards of 100 kg oligonucleotide per year and requires a relatively modest initial investment. The current cost of goods for aptamer synthesis at the kilogram scale is estimated at $500 /g, comparable to that for highly optimized antibodies. Continuing improvements in process development are expected to lower the cost of goods to <$100/g in five years.
5) Stability. Therapeutic aptamers are chemically robust. They are intrinsically adapted to regain activity following exposure to heat, denaturants, etc. and can be stored for extended periods (>1 yr) at room temperature as lyophilized powders. In contrast, antibodies must be stored refrigerated.
The human immunodeficiency virus (HIV), the cause of acquired immunodeficiency syndrome (AIDS), remains an extremely serious threat to public health worldwide. Globally, over 40 million people are infected with HIV, with roughly 14,000 new infections arising each day (UNAIDS Report, 2002). Clearly, the best long-term solution for controlling the AIDS epidemic is development of a safe and effective HIV vaccine. The gp120 subunit is the primary viral antigen against which humoral immune responses are mounted (Profy, 1990; reviewed in Poignard et al., 2001). The mature envelope glycoprotein exists as a trimer that arises through processing of a larger precursor (gp160) to gp120 and gp41 components which non-covalently associate on the virion surface (Kowalski, et al., 1987; Lu et al., 1995; Burton, 1997). Each gp120 monomer consists of five constant regions (C1-C5) interspersed with five variable regions (V1-V5) (Starcich et al., 1986). Variable regions tend to be oriented on the outer surface of the protein where they help to shield core regions from immune surveillance. Gp120 is also heavily glycosylated (Leonard, 1990). The surface variability and glycosylation of gp120 reduce its immunogenicity. Though progress is being made in development of vaccines that stimulate cell-mediated immune responses, induction of an effective neutralizing antibody response by an HIV vaccine candidate in a clinical setting remains an urgent and unmet medical need.
Current opinion among researchers on the most efficacious route to HIV vaccine development centers on the need to induce both humoral and cell-mediated immune responses that include broadly neutralizing antibodies, and cytotoxic T-lymphocytes (CTL) and T-helper responses. The CTL cells are CD8+ and the T-helper cells are CD4+. However, vaccine-induced neutralizing antibody responses in clinical trials to date have been weak and ineffective against primary viruses.
Much recent effort has been invested in development of gp120 subunit vaccines (reviewed in Graham, 2002). However, antibodies generated against monomeric gp120 are generally not neutralizing, or at best, are capable only of neutralizing laboratory-adapted strains of HIV (Belshe et al., 1994; Kahn, et al., 1994) and not the more medically-relevant, primary HIV type 1 (HIV-1) isolates (Cohen, 1994). However, passive antibody studies in nonhuman primate models have shown that neutralizing antibodies do in fact protect against infection (Prince et al., 1991; Putkonen, P. et al., 1991; Emini et al., 1992). Indeed, antibody is the sole immune component that can neutralize virus prior to entry, unlike CTLs which are effective only after establishment of cellular infection. Induction of an effective neutralizing antibody response by a gp120-derived immunogen remains an elusive goal.
Variability of the envelope glycoprotein plays a key role in the exceptional ability of HIV to avoid immune attack. Viral mutations accumulate readily as infection progresses, generating a diverse population of variants, even within a single infected individual, and providing opportunities for escape from CTL control (Gaschen et al., 2002). This diversity presents significant challenges to vaccine design. Together, surface variability and extensive glycosylation contribute to the relatively poor immunogenicity of monomeric gp120 immunogens (Leonard, 1990; Langlois et al., 1998; Kwong et al., 2002; Wei et al., 2003). Interestingly, recent results have shown that infected individuals can and often do generate neutralizing antibody responses. Unfortunately these responses appear to lag behind the rapid evolution of the env gene and are thus unable to resist and clear the high level viremia associated with a productive infection (Wei et al., 2003 and Richman et al., 2003). These results do suggest however that individuals vaccinated with appropriate immunogens may be able to generate an immune response capable of protecting against the relatively low viral loads associated with initial exposure to HIV.
A variety of strategies have been developed in pursuit of effective immune responses to HIV, with testing of immunogens in a number of clinical trials (reviewed in Emini, 2002; Graham, 2002). Live-attenuated HIV vaccines have shown potential to induce protection in nonhuman primates (Nixon et al., 1999). However, safety concerns have largely directed current efforts away from use of live-attenuated and whole-killed viral vaccines. Subunit vaccines, like those used in the recent Vaxgen trial, based on HIV surface proteins (primarily gp120 or gp 160) though safe and generally well-tolerated, have not succeeded in eliciting neutralizing antibody responses across populations (Wantanabe, 2003). Neutralizing antibody responses against laboratory-adapted HIV strains produced by most subunit vaccines are several-fold lower than those seen during HIV-1 infection (Graham et al., 2002). Type-specific neutralization can sometimes be achieved, usually corresponding to the origin of the vaccine antigen. However, neutralization of primary R5 HIV isolates has not been observed (Mascola et al., 1996). Alternative vaccine concepts being evaluated in clinical trials include vectored and DNA vaccines that rely on antigen production within cells and surface display on MHC class I molecules. Emerging evidence suggests that durable CD8+CTL activity can be induced using these approaches (Graham et al., 2002). However, as noted above, CTL-based mechanisms succeed only in eradicating cells that have already become infected. While potentially able to control viral load and attenuate disease, cell-mediated mechanisms alone are unlikely to prevent HIV infection.
Potent and enduring neutralizing antibodies are a critical component of any vaccine-induced immunity. Recently efforts have been made to design better gp120 based immunogens based upon the stabilization of conformations of gp120 known to expose neutralizing epitopes that are normally exposed only transiently during infection. The HIV entry process is complex, involving a sequence of protein-protein contacts choreographed by gp120. HIV binding interactions with CD4 receptor and with CCR5/CXCR4 co-receptors (FIG. 2) each appear to be accompanied by significant structural rearrangement in gp120 (Doranz et al., 1997). Initial binding of CD4 induces a conformational change in gp120 through shifting of variable loops V1 and V2 (FIG. 3), thereby exposing conserved gp120 core residues that comprise the chemokine co-receptor binding site (Wu et al., 1996; Trkola et al., 1996). CD4-inducible (CD4i) antibodies recognizing this unmasked core region (17b, 48d) are reported to have neutralizing activity (Thali et al., 1993; Sullivan et al., 1998). Subsequent binding of gp120 to either CCR5 or CXCR4 stimulates a second conformational shift in gp 120 that enables exposure of the fusion domain of gp41 responsible for fusion of viral and cellular membranes. In one study relying on the conformational changes associated with the HIV entry process, strong neutralizing antibody responses were generated in rhesus macaques using a covalently crosslinked gp120/CD4 complex as an immunogen (Fouts et al., 2002). Unfortunately a significant portion of this effect is likely mediated by anti-CD4 antibody responses. Another recent advance has been in the area of CD4 mimics. Using a scyllatoxin scaffold Martin et al. have engineered a small mini-protein that can functionally mimic that action of CD4 on gp120 (Martin et al., 2003). They propose one use of this mini-protein to be as an immunogen that in conjunction with gp120 will expose the highly conserved CD4-inducible (CD4i) epitope which is normally occluded in the absence of CD4 receptor.
Several lines of biochemical and structural evidence support CD4 binding-induced structural changes in gp120, including: increased protease sensitivity of gp120 variable region loops (Sattentau et al., 1991), as well as CD4-stimulated accessibility of the chemokine receptor binding site (Sattentau et al., 1993; Wu, et al., 1996) and of epitopes for antibodies that compete for co-receptor binding (Thali et al., 1993; Zhang et al., 1999). Recent thermodynamic analysis of gp120/CD4/MAb interactions revealed unusually high changes in entropy upon CD4 binding offering further support for the hypothesis that gp120 undergoes a major conformational change upon receptor binding (Kwong et al., 2002). Structural analysis of the ternary complex of CD4 and gp120 with CD4i neutralizing antibody 17b confirmed that stabilizing interactions with CD4 play a significant role in exposure or formation of the CCR5 binding region (Kwong et al., 1998).
Receptor and co-receptor binding sites are attractive targets for use in vaccine design or for therapeutic intervention as they show conservation among different HIV subtypes and must be exposed on the gp120 surface, at least transiently, in order for the virus to gain entry into cells. The CCR5 binding region, in particular, is one of the most highly conserved surfaces on the gp120 core (Rizzuto et al., 1998). Antibody responses to highly conserved epitopes, integral to the fundamental mechanism of HIV entry, are expected to show neutralizing activity even against diverse HIV subtypes. Thus, there is a need for a preventative, prophylactic agent that can bind specifically to gp120 and induce a conformational change that reveals suitable immunogenic epitopes and results in a humoral immune response to prevent or treat infection of cells by HIV.